Chemiluminescent acridinium compounds have emerged to be extremely useful labels for immunoassays and nucleic acid assays. Hydrophilic acridinium esters containing N-sulfopropyl groups (NSP) are useful for protein labeling as well as for the preparation of small molecule conjugates, as described by Law et al. in U.S. Pat. No. 5,656,426 and Natrajan et al. in U.S. Pat. No. 6,664,043, the disclosures of which are hereby incorporated by reference. These hydrophilic acridinium esters offer improved performance in immunoassays.
The synthesis of acridinium esters containing N-sulfopropyl groups entails N-alkylation of the corresponding acridine esters with the potent carcinogen 1,3-propane sultone. In general, this reaction is carried out by heating the acridine ester with a vast excess of propane sultone in the absence of solvent, as described by Law et al in U.S. Pat. No. 5,656,426, incorporated by reference herein. This reaction is illustrated in FIG. 1. The reaction as described by Law for the synthesis of NSP-DMAE-NHS ester (FIG. 1, R=NHS) suffers from several drawbacks that include a relatively low yield of isolated product (25%) and the need to use a vast excess of propane sultone (50-fold) over the acridine precursor.
As reported recently by Bolt and Golka in Toxicology Lett. 2004, 151, 251-254, the disclosure of which is incorporated by reference, propane sultone is directly alkylating, genotoxic and carcinogenic. Bolt and Golka observe that “malignancies observed within a group of persons exposed to 1,3-propane sultone appear surprisingly consistent with the expectations from the available animal studies” and are characterized by latency times up to 30-40 years or more after limited periods of past exposure. Thus, chemical transformations under reaction conditions that limit the use this toxic reagent are desirable.
More recently, Evangelista et al. in U.S. Pat. No. 6,924,154, the disclosure of which is hereby incorporated by reference, reported the synthesis of an acridinium ester with an N-sulfopropyl group by microwave heating of the corresponding acridine ester and a 20-fold excess of neat 1,3-propane sultone. This synthetic process required a second step to hydrolyze polysulfonated acridinium esters formed in the reaction with strong acid to convert them to the monosulfonate N-sulfopropyl group, followed by extensive chromatographic purification to afford an overall yield of 53% of the N-sulfopropyl-containing acridinium ester. Clearly, if the acridine ester contains acid-sensitive functional groups, such as the NHS ester, the acid hydrolysis step cannot be performed and the overall yield of the desired monosulfonate product is likely to be quite low, as observed by Law in U.S. Pat. No. 5,656,426. Moreover, the necessity for extensive chromatographic purification of the product is also undesirable.
Adamczyk et al. in J. Org. Chem. 1998, 63, 5636-5639, the disclosure of which is hereby incorporated by reference, reported that the introduction of the N-sulfopropyl group in acridine sulfonamides can be performed using the reagent neopentyl 3-trifloxypropane sulfonate. Although this procedure obviates using 1,3-propane sultone, the reagent 3-trifloxypropane sulfonate had to be synthesized separately and moreover, N-alkylation using this reagent required 7 days to afford 34% conversion to product.
The above cited literature points to the general difficulty in introducing the N-sulfopropyl group in acridine compounds which necessitates conducting the reaction in neat alkylating reagent, such as propane sultone, at elevated temperatures followed by acid treatment to hydrolyze polysulfonated material and subsequent extensive chromatographic purification of the desired N-sulfopropyl acridinium ester. Clearly, a suitable and relatively benign reaction medium that would be conducive to achieving this chemical transformation in good yields but using only limited quantities of propane sultone and without the need for extensive chromatographic purification would be beneficial. Moreover, any process that minimizes polysulfonate formation, would permit the N-alkylation of acridine esters containing acid sensitive functional groups.
In recent years, ionic liquids (ILs) have becoming increasingly popular for conducting chemical reactions and are commonly referred to as “green solvents” not only because they can be recycled but, also because of their extremely low volatility, low toxicity, non-flammability, high thermal stability and, their ability to dissolve a wide range of solutes. A review by Holbrey and Seddon in Clean Products and Processes 1999, 1, 223-236, the disclosure of which is hereby incorporated by reference, describes some of the properties of ionic liquids. Another review by Hagiwara and Ito in J. Fluorine Chem. 2000, 105, 221-227, incorporated by reference herein, describes room temperature ionic liquids of alkylimidazolium cations and fluoroanions. Hagiwara and Ito state that “a room temperature ionic liquid, or molten salt, is defined as a material containing only ionic species without any neutral molecules and having a melting point of less than 298 K.” Many ionic liquids derived from alkylimidazolium cations are commercially available and two in particular that have been used in the literature are 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] and 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4].
Ionic liquids have been explored as reaction media for various synthetic organic transformations including oxidations as described by Earle et al. in Org. Lett. 2004, 6, 707-710; Heck reaction as described by Mo et al. in J. Am. Chem. Soc. 2005, 127, 751-760; N-alkylation of pyrroles and indoles as described by Jorapur et al. in Tetrahedron Lett. 2006, 47, 2435-2438; N-alkylation of phthalimides as described by Le et al. in Synthesis 2004, 2, 208-212; and peptide synthesis as described by Miao and Chan in J. Org. Chem. 2005, 70, 3251-3255, the disclosures of which are hereby incorporated by reference. U.S. Patent Pub. 2007/0142690 to Elomari, the disclosure of which is hereby incorporated by reference, discloses alkylation of olefins with isoparaffins in ionic liquids. The use of ILs as reaction media for the N-alkylation of acridine esters to N-sulfopropyl acridinium esters has not been reported in the literature.
While ionic liquids have found utility in a variety of synthetic transformations, their use to date has been rather limited, particularly with regard to commercial scale syntheses.